Rice Cultivar L-206

ABSTRACT

A rice cultivar designated L-206 is disclosed. The invention relates to the seeds of rice cultivar L-206, to the plants of rice L-206 and to methods for producing a rice plant produced by crossing the cultivar L-206 with itself or another rice variety. The invention further relates to hybrid rice seeds and plants produced by crossing the cultivar L-206 with another rice cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive rice cultivar,designated L-206. All publications cited in this application are hereinincorporated by reference.

Rice is an ancient agricultural crop and is today one of the principalfood crops of the world. There are two cultivated species of rice: Oryzasaliva L., the Asian rice, and O. glaberrima Steud., the African rice.O. sativa L. constitutes virtually all of the world's cultivated riceand is the species grown in the United States. Three major riceproducing regions exist in the United States: the Mississippi Delta(Arkansas, Mississippi, northeast Louisiana, southeast Missouri), theGulf Coast (southwest Louisiana, southeast Texas), and the CentralValleys of California.

Rice is a semi-aquatic crop that benefits from flooded soil conditionsduring part or all of the growing season. In the United States, rice isgrown on flooded soils to optimize grain yields. Heavy clay soils orsilt loam soils with hard pan layers about 30 cm below the surface aretypical rice-producing soils because they minimize water losses fromsoil percolation. Rice production in the United States can be broadlycategorized as either dry-seeded or water-seeded. In the dry-seededsystem, rice is sown into a well-prepared seed bed with a grain drill orby broadcasting the seed and incorporating it with a disk or harrow.Moisture for seed germination is from irrigation or rainfall. For thedry-seeded system, when the plants have reached sufficient size (four-to five-leaf stage), a shallow permanent flood of water 5 to 16 cm deepis applied to the field for the remainder of the crop season.

In the water-seeded system, rice seed is soaked for 12 to 36 hours toinitiate germination, and the seed is broadcast by airplane into aflooded field. The seedlings emerge through a shallow flood, or thewater may be drained from the field for a short period of time toenhance seedling establishment. A shallow flood is maintained until therice approaches maturity. For both the dry-seeded and water-seededproduction systems, the fields are drained when the crop is mature, andthe rice is harvested 2 to 3 weeks later with large combines. In ricebreeding programs, breeders try to employ the production systemspredominant in their respective region. Thus, a drill-seeded breedingnursery is used by breeders in a region where rice is drill-seeded and awater-seeded nursery is used in regions where water-seeding isimportant.

Rice in the United States is classified into three primary market typesby grain size, shape, and chemical composition of the endosperm:long-grain, medium grain and short-grain. Typical U.S. long-graincultivars cook dry and fluffy when steamed or boiled, whereas medium-and short-grain cultivars cook moist and sticky. Long-grain cultivarshave been traditionally grown in the southern states and generallyreceive higher market prices.

Although specific breeding objectives vary somewhat in the differentregions, increasing yield is a primary objective in all programs. Grainyield of rice is determined by the number of panicdes per unit area, thenumber of fertile florets per panicle, and grain weight per floret.Increases in any or all of these yield components may provide amechanism to obtain higher yields. Heritable variation exists for all ofthese components, and breeders may directly or indirectly select forincreases in any of them.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to lowtemperatures, and better agronomic characteristics on grain quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection, ora combination of these methods.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from 8 to 12 years from the time the firstcross is made and may rely on the development of improved breeding linesas precursors. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of rice plant breeding is to develop new, unique and superiorrice cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by self-pollination and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same rice traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new rice cultivars.

The development of new rice cultivars requires the development andselection of rice varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as semi-dwarf plant type, pubescence, awns, and apiculuscolor which indicate that the seed is truly a hybrid. Additional data onparental lines, as well as the phenotype of the hybrid, influence thebreeders decision whether to continue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, rice breeders commonly harvest one or moreseeds from each plant in a population and thresh them together to form abulk. Part of the bulk is used to plant the next generation and part isput in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh panicles with a machine than to removeone seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Rice, Oryza sativa L., is an important and valuable field crop. Thus, acontinuing goal of plant breeders is to develop stable, high yieldingrice cultivars that are agronomically sound. The reasons for this goalare obviously to maximize the amount of grain produced on the land usedand to supply food for both animals and humans. To accomplish this goal,the rice breeder must select and develop rice plants that have thetraits that result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related are will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, no limiting in scope. In variousembodiments, one or more of the above described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel rice cultivardesignated L-206. This invention thus relates to the seeds of ricecultivar L-206, to the plants of rice L-206 and to methods for producinga rice plant produced by crossing the rice L-206 with itself or anotherrice line.

Thus, any such methods using the rice variety L-206 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using rice variety L-206as a parent are within the scope of this invention. Advantageously, therice variety could be used in crosses with other, different, rice plantsto produce first generation (F₁) rice hybrid seeds and plants withsuperior characteristics.

In another aspect, the present invention provides for single geneconverted plants of L-206. The single transferred gene may preferably bea dominant or recessive allele. Preferably, the single transferred genewill confer such traits as herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility, malesterility, enhanced nutritional quality, and industrial usage. Thesingle gene may be a naturally occurring rice gene or a transgeneintroduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of rice plant L-206. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing rice plant, and ofregenerating plants having substantially the same genotype as theforegoing rice plant. Preferably, the regenerable cells in such tissuecultures will be embryos, protoplasts, meristematic cells, callus,pollen, leaves, anthers, root tips, flowers, seeds, panicles or stems.Still further, the present invention provides rice plants regeneratedfrom the tissue cultures of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Alkali Spreading Value. Indicator of gelatinization temperature and anindex that measures the extent of disintegration of milled rice kernelin contact with dilute alkali solution. Standard long grains have 3 to 5Alkali Spreading Value (intermediate gelatinization temperature).Standard medium and short grain rice have 6 to 7 Alkali Spreading Values(low gelatinization temperature).

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Apparent Amylose Percent. The most important grain characteristic thatdescribes cooking behavior in each grain class, or type, i.e., long,medium and short grain. The percentage of the endosperm starch of milledrice that is amylose. Standard long grains contain 20 to 23% amylose.Rexmont type long grains contain 24 to 25% amylose. Short and mediumgrain rice contain 16 to 19% amylose. Waxy rice contains 0% amylose.Amylose values will vary over environments.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Blanking %. Visual estimate of the percent of sterile florets (floretsthat are empty with no filled kernels) in the panicle as a measurementof cool temperature induced pollen sterility. This data may be collectedin screening nurseries at cool locations, cool years, and also inscreening tests in refrigerated greenhouses.

Days to 50% heading. Average number of days from planting to the daywhen 50% of all panicdes are exerted at least partially through the leafsheath. A measure of maturity.

Elongation. Cooked kernel elongation is the ratio of the cooked kernellength divided by the uncooked kernel length. Extreme cooked kernelelongation is a unique feature of basmati type rice and an importantquality criteria for that market type.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the cultivar, except for the characteristics derivedfrom the converted gene.

Grain Length (L). Length of a rice grain is measured in millimeters.

Grain Width (W). Width of a rice grain is measured in millimeters.

Grain Yield. Grain yield is measured in pounds per acre and at 14.0%moisture. Grain yield of rice is determined by the number of paniclesper unit area, the number of fertile florets per panicle, and grainweight per floret.

Harvest Moisture. The percent of moisture of the grain when harvested.

Length/Width (LAW) Ratio. This ratio is determined by dividing theaverage length (L) by the average width (W).

Lodging Resistance (also called Straw Strength). Lodging is measured asa subjective rating and is percentage of the plant stems leaning orfallen completely to the ground before harvest. Relative scale.

1000 Grain Wt. The weight of 1000 rice grains as measured in grams. Itcan be for paddy, brown or milled rice.

Plant Height. Plant height in centimeters is taken from soil surface tothe tip of the extended panicle at harvest.

Peak Viscosity. The maximum viscosity attained during heating when astandardized instrument-specific protocol is applied to a defined riceflour-water slurry.

Trough Viscosity. The minimum viscosity after the peak, normallyoccurring when the sample starts to cool.

Final Viscosity. Viscosity at the end of the test or cold paste

Breakdown. The peak viscosity minus the hot paste viscosity.

Setback. Setback 1 is the final viscosity minus trough viscosity.Setback 2 is the final viscosity minus peak viscosity and is what ismost commonly referred to for rice quality testing.

RVA Viscosity. Rapid Visco Analyzer is a widely used laboratoryinstrument to examine paste viscosity, or thickening ability of milledrice during the cooking process.

RVU. The RVA scale is measured in RVUs. This is the native viscosityunit of the RVA. 1 RVU is equivalent to 12 CP. CP equals “centipoises”which equals unit of viscosity (kg s⁻¹ m⁻¹) and 1 kg s⁻¹ m⁻¹ equals 1000centipoises.

Hot Paste Viscosity. Viscosity measure of rice flour/water slurry afterbeing heated to 95° C. Lower values indicate softer and stickier cookingtypes of rice.

Cool Paste Viscosity. Viscosity measure of rice flour/water slurry afterbeing heated to 95° C. and uniformly cooled to 50° C. (AmericanAssociation of Cereal Chemist). Values less than 200 for cool pasteindicate softer cooking types of rice.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

Texture Score. A relative subjective score used by the breeder inevaluating cooked rice samples. A score of 4 being most sticky and ascore of 2 being the least sticky.

DETAILED DESCRIPTION OF THE INVENTION

Rice cultivar L-206 is a very early to early maturing, glabrous,semidwarf, long-grain, photoperiod insensitive rice variety. Ricecultivar L-206 has improved cooking quality and the earlier maturity ofthis cultivar favors its production in more regions including the coolerrice growing area of California.

The cultivar has shown uniformity and stability as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity.

Rice Cultivar L-206 has the following morphologic and othercharacteristics (based primarily on data collected in California).

TABLE 1 VARIETY DESCRIPTION INFORMATION Rice Grain type: Long Days tomaturity (50% heading): 82 Culm (Degrees from perpendicular afterflowering) Angle: Erect (less than 30° C.) Length: 86.0 cm Height Class:Semidwarf Internode Color (after flowering) Green Strength (lodgingresistance) Strong (no lodging) Flag Leaf (After Heading) Length: 30.4cm Width: 1.63 cm Pubescence: Glabrous Leaf Angle (After heading): ErectBlade Color: Green Ligule Length (from base of collar 1.35 cm to thetip, at late vegetative stage): Color (late vegetative stage): WhiteShape: 2-Cleft Collar Color Pale Green (late vegetative stage): AuricleColor Pale Green (late vegetative stage): Panicle Length: 19.6 cm Type:Intermediate Secondary Branching: Heavy Exsertion (near maturity): Lessthan 90% Axis: Straight Shattering: Very Low (Less than 1%)Threshability: Intermediate Grain (Spikelet) Awns (After full heading):Absent Apiculus Color (at maturity): Red Stigma Color: Light green Lemmaand Palea Color Straw (at maturity): Lemma and Palea Pubescence: Hairson Lemma Keel Spikelet Sterility (at maturity): Highly fertile (greaterthan 90%) Grain (Seed) Seed Coat Color: Light Brown Endosperm Type:Nonglutinous (nonwaxy) Endosperm Translucency: Clear EndospermChalkiness: Small (less than 10% of sample) Scent: Nonscented ShapeClass (Length/width ratio): Paddy: Long (3.4:1 and more) Length: 10.4 mmWidth: 2.5 mm L/W ratio: 4.2 1000 Grains: 28.4 g Brown: Long (3.1:1 andmore) Length: 8.2 mm Width: 2.2 mm L/W ratio: 3.7 1000 Grains: 23.3 gMilled: Long (3.0:1 and more) Length: 7.3 mm Width: 2.1 mm L/W ratio:3.5 1000 Grains: 20.8 g Milling Quality (% hulls): 21.0 Milling Yield (%while kernel 62.8 (head) rice to rough rice): % Amylose: 22.8 AlkaliSpreading Value 3–5 (1.7% KOH Solution) Gelatinization Temperature Type:Intermediate Amylographic Paste Viscosity (RVA measured in RVU): Peak:252 Hot Paste: 161 Cooled Paste: 298 “Breakdown” “Setback”: 91/46Resistance to Low Temperature Germination and Seedling Vigor: MediumFlowering (spikelet fertility): Medium Seedling Vigor Not Related to LowTemperature Vigor: Medium Disease Resistance Rice Blast (Pyriculariaoryzae): Susceptible to California Race IG1 Aggregate Sheath SpotIntermediate: (Rhizoctonia oryzae-sativae): in between ModeratelyResistant and Moderately Susceptible Stem Rot (Sclerotium oryzae):Intermediate: in between Moderately Resistant and Moderately SusceptibleInsect Resistance: Rice Water Weevil Susceptible (Lissorhoptrusoryzophilus)

This invention also is directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plantwherein either the first or second parent rice plant is a rice plant ofthe line L-206. Further, both first and second parent rice plants cancome from the rice cultivar L-206. Still further, this invention also isdirected to methods for producing a rice cultivar L-206-derived riceplant by crossing rice cultivar L-206 with a second rice plant andgrowing the progeny seed, and repeating the crossing and growing stepswith the rice cultivar L-206-derived plant from 0 to 7 times. Thus, anysuch methods using the rice cultivar L-206 are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using rice cultivar L-206 as a parent arewithin the scope of this invention, including plants derived from ricecultivar L-206. Advantageously, the rice cultivar is used in crosseswith other, different, rice cultivars to produce first generation (F₁)rice seeds and plants with superior characteristics.

It should be understood that the cultivar can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which rice plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, glumes,panicles, leaves, stems, roots, root tips, anthers, pistils and thelike.

Rice cultivar L-206 is most similar to rice cultivar L-204. Ricecultivar L-206 has improved cooking quality. Rice cultivar L-206 has asignificantly earlier maturity than either rice cultivars L-204 andL-205 (L-206 matures 6 days earlier than L-205). Rice cultivar L-206 hasa higher setback value than L-204, indicating less sticky cooked graintexture. In addition, rice cultivar L-206 has a culm length that is 6 cmshorter than the culm length of rice cultivar L-205.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, rice is transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation”, inMethods in Plant Molecular Biology & Biotechnology in Glich, et al.,(Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors andGUS gene cassettes are available from Clone Tech Laboratories, Inc.,Palo Alto, Calif. while luciferase expression vectors and luciferasegene cassettes are available from Pro Mega Corp. (Madison, Wis.).General methods of culturing plant tissues are provided for example byMaki, et al., “Procedures for Introducing Foreign DNA into Plants” inMethods in Plant Molecular Biology & Biotechnology, Glich, et al., (Eds.pp. 67-88 CRC Press, 1993); and by Phillips, et al., “Cell-TissueCulture and In-Vitro Manipulation” in Corn & Corn Improvement, 3rdEdition; Sprague, et al., (Eds. pp. 345-387 American Society of AgronomyInc., 1988). Methods of introducing expression vectors into plant tissueinclude the direct infection or co-cultivation of plant cells withAgrobacterium tumefaciens, described for example by Horsch et al.,Science, 227:1229 (1985). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber, et al., supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using a microprojectile media delivery system with a biolisticdevice or using Agrobacterium-mediated transformation. Transformantplants obtained with the protoplasm of the invention are intended to bewithin the scope of this invention.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed rice plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the rice plant(s).

Expression Vectors for Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN, p. 1-4(1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in rice. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in rice. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in rice or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in rice.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in rice.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in rice. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is rice. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant cultivar can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvishikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fafty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content, 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene; 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved inrice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S.Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Additionally,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of rice target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic cultivar. The transgenic cultivar could then becrossed, with another (non-transformed or transformed) cultivar, inorder to produce a new transgenic cultivar. Alternatively, a genetictrait which has been engineered into a particular rice cultivar usingthe foregoing transformation techniques could be moved into anothercultivar using traditional backcrossing techniques that are well knownin the plant breeding arts. For example, a backcrossing approach couldbe used to move an engineered trait from a public, non-elite cultivarinto an elite cultivar, or from a cultivar containing a foreign gene inits genome into a cultivar which does not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Single Gene Conversion

When the term rice plant is used in the context of the presentinvention, this also includes any single gene conversions of thatcultivar. The term single gene converted plant as used herein refers tothose rice plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a cultivar are recovered inaddition to the single gene transferred into the cultivar via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thecultivar. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental rice plants,the recurrent parent, for that cultivar, i.e., backcrossing 1, 2, 3, 4,5, 6, 7, 8, 9 or more times to the recurrent parent. The parental riceplant which contributes the gene for the desired characteristic istermed the nonrecurrent or donor parent. This terminology refers to thefact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental rice plant to whichthe gene or genes from the nonrecurrent parent are transferred is knownas the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehiman & Sleper, 1994; Fehr, 1987). In atypical backcross protocol, the original cultivar of interest (recurrentparent) is crossed to a second cultivar (nonrecurrent parent) thatcarries the single gene of interest to be transferred. The resultingprogeny from this cross are then crossed again to the recurrent parentand the process is repeated until a rice plant is obtained whereinessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent as determined at the 5% significance level when grown in the sameenvironmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original cultivar. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of rice and regenerationof plants therefrom is well known and widely published. For example,reference may be had to Komatsuda, T. et al., Crop Sci. 31:333-337(1991); Stephens, P. A., et al., Theor. Appl. Genet. (1991) 82:633-635;Komatsuda, T. et al., Plant Cell, Tissue and Organ Culture, 28:103-113(1992); Dhir, S. et al., Plant Cell Reports (1992) 11:285-289; Pandey,P. et al., Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al., PlantScience 81:245-251 (1992); as well as U.S. Pat. No. 5,024,944 issuedJun. 18, 1991 to Collins et al., and U.S. Pat. No. 5,008,200 issued Apr.16, 1991 to Ranch et al. Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce rice plantshaving the physiological and morphological characteristics of ricevariety L-206.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which rice plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as pollen, flowers, embryos, ovules,seeds, pods, leaves, stems, pistils, anthers and the like. Thus, anotheraspect of this invention is to provide for cells which upon growth anddifferentiation produce a cultivar having essentially all of thephysiological and morphological characteristics of L-206.

The present invention contemplates a rice plant regenerated from atissue culture of a variety (e.g., L-206) or hybrid plant of the presentinvention. As is well known in the art, tissue culture of rice can beused for the in vitro regeneration of a rice plant. Tissue culture ofvarious tissues of rice and regeneration of plants therefrom is wellknown and widely published. For example, reference may be had to Chu, Q.R., et al., (1999) “Use of bridging parents with high antherculturability to improve plant regeneration and breeding value in rice”,Rice Biotechnology Quarterly 38:25-26; Chu, Q. R., et al., (1998), “Anovel plant regeneration medium for rice anther culture of Southern U.S.crosses”, Rice Biotechnology Quarterly 35:15-16; Chu, Q. R., et al.,(1997), “A novel basal medium for embryogenic callus induction ofSouthern US crosses”, Rice Biotechnology Quarterly 32:19-20; and Oono,K., “Broadening the Genetic Variability By Tissue Culture Methods”, Jap.J. Breed. 33 (Suppl.2), 306-307, illus. 1983. Thus, another aspect ofthis invention is to provide cells which upon growth and differentiationproduce rice plants having the physiological and morphologicalcharacteristics of variety L-206.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both cultivars and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two cultivars.Other published reports also indicated that “non-traditional” tissuesare capable of producing somatic embryogenesis and plant regeneration.K. P. Rao et al., Maize Genetics Cooperation Newsletter, 60:64-65(1986), refers to somatic embryogenesis from glume callus cultures andB. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987) indicatessomatic embryogenesis from the tissue cultures of corn leaf segments.Thus, it is clear from the literature that the state of the art is suchthat these methods of obtaining plants are routinely used and have avery high rate of success.

Tissue culture of corn is described in European Patent Application,publication 160,390. Corn tissue culture procedures are also describedin Green and Rhodes, “Plant Regeneration in Tissue Culture of Maize,”Maize for Biological Research (Plant Molecular Biology Association,Charlottesville, Va. 367-372, (1982)) and in Duncan et al., “TheProduction of Callus Capable of Plant Regeneration from Immature Embryosof Numerous Zea Mays Genotypes,” 165 Planta 322:332 (1985). Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce corn plants having the physiological andmorphological characteristics of rice cultivar L-206.

The utility of rice cultivar L-206 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae.

This invention also is directed to methods for producing a rice plant bycrossing a first parent rice plant with a second parent rice plantwherein the first or second parent rice plant is a rice plant of thevariety L-206. Further, both first and second parent rice plants cancome from the rice variety L-206. Thus, any such methods using the ricevariety L-206 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing rice variety L-206 as a parent are within the scope of thisinvention, including those developed from varieties derived from ricevariety L-206. Advantageously, the rice variety could be used in crosseswith other, different, rice plants to produce the first generation (F₁)rice hybrid seeds and plants with superior characteristics. The varietyof the invention can also be used for transformation where exogenousgenes are introduced and expressed by the variety of the invention.Genetic variants created either through traditional breeding methodsusing variety L-206 or through transformation of L-206 by any of anumber of protocols known to those of skill in the art are intended tobe within the scope of this invention.

The following describes breeding methods that may be used with cultivarL-206 in the development of further rice plants. One such embodiment isa method for developing an L-206 progeny rice plant in a rice plantbreeding program comprising: obtaining the rice plant, or a partthereof, of cultivar L-206 utilizing said plant or plant part as asource of breeding material and selecting an L-206 progeny plant withmolecular markers in common with L-206 and/or with morphological and/orphysiological characteristics selected from the characteristics listedin Tables 2 or 3. Breeding steps that may be used in the rice plantbreeding program include pedigree breeding, back crossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example SSR markers) and the making of double haploidsmay be utilized.

Another method involves producing a population of cultivar L-206 progenyrice plants, comprising crossing cultivar L-206 with another rice plant,thereby producing a population of rice plants, which, on average, derive50% of their alleles from cultivar L-206. A plant of this population maybe selected and repeatedly selfed or sibbed with a rice cultivarresulting from these successive filial generations. One embodiment ofthis invention is the rice cultivar produced by this method and that hasobtained at least 50% of its alleles from cultivar L-206.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes rice cultivarL-206 progeny rice plants comprising a combination of at least two L-206traits selected from the group consisting of those listed in Tables 1,2, 3, and 4 or the L-206 combination of traits listed in the Summary ofthe Invention, so that said progeny rice plant is not significantlydifferent for said traits than rice cultivar L-206 as determined at the5% significance level when grown in the same environment. Usingtechniques described herein, molecular markers may be used to identifysaid progeny plant as a L-206 progeny plant. Mean trait values may beused to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions. Once such a variety is developed its value issubstantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance, and plant performance in extremeenvironmental conditions.

Progeny of cultivar L-206 may also be characterized through their filialrelationship with rice cultivar L-206, as for example, being within acertain number of breeding crosses of rice cultivar L-206. A breedingcross is a cross made to introduce new genetics into the progeny, and isdistinguished from a cross, such as a self or a sib cross, made toselect among existing genetic alleles. The lower the number of breedingcrosses in the pedigree, the closer the relationship between ricecultivar L-206 and its progeny. For example, progeny produced by themethods described herein may be within 1, 2, 3, 4 or 5 breeding crossesof rice cultivar L-206.

The seed of rice cultivar L-206, the plant produced from the cultivarseed, the hybrid rice plant produced from the crossing of the cultivar,hybrid seed, and various parts of the hybrid rice plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Tables

In Table 2, agronomic and milling characteristics from six years oftesting at the Rice Experimental Station are shown for the presentinvention and two other rice cultivars. The data is part of the veryearly group of the multiple location University of CaliforniaCooperative Extension Statewide Yield Testing program that was conductedduring 2000 to 2005. Column 1 shows the characteristic; column 2 showsthe year of the test; column 3 shows the results for rice cultivarL-206; column 4 shows the results for rice cultivar L-204; and column 5shows the results for rice cultivar L-205. The Seedling vigor score is avisual score where a score of 1 is poor and a score of 5 is excellent.The Blanking percent was taken in a greenhouse. The Stem Rot score israted where a score of zero indicates no damage and a score of 10indicates that the plant has been killed.

TABLE 2 Characteristic Year L-206 L-204 L-205 Seedling Vigor 2003 4.7  4.9*   4.8 Score 2004 4.6   4.8*   4.7 2005 4.7   4.8   4.7 Days to50% 2003 73   77*   77* Heading 2004 79   84*   85* 2005 76   79*   84*Plant Height (cm) 2003 85   85   92* 2004 86   87   99* 2005 91   90  94 Lodging (%) 2003 28   1   36 2004 28   1*   56* 2005 25   1   6Grain Yield (14% 2003 9340  9480  9370 moisture, lb/acre) 2004 1093010830 10350 2005 8400  8140  8920 Head Rice Yield 2003 65   69*   66 (%)2004 65   65   64 2005 60   64*   63* Blanking (%) 2003 5   20*   7(Greenhouse) 2004 5   8   8 2005 15   15   8* Stem Rot Score 2003 6.1  5.9   5.2* 2004 9.1   7.2*   8.7 2005 6.3   6.5   5.9* *Significantlydifferent from L-206 at 0.05 probability level

Table 3 shows the physicochemical characteristics for the presentinvention and 2 other rice cultivars from the Rice Experimental Stationin Biggs, Calif. Laboratory analysis of milled rice samples was providedby the USDA-ARS Rice Research Unit in Beaumont, Tex., using standardphysicochemical tests. Column 1 shows the year of the test; column 2indicates the cultivar and columns 3-6 are the values for the RapidVisco Analyzer where the peak viscosity, the hot paste viscosity, thecool paste viscosity and the set back values are all listed. Column 7shows the amylose percent; column 8 shows the alkali spreading value andcolumn 9 shows the subjective texture score.

TABLE 3 RVA Viscosity (RVU) Hot Cool Alkali Texture Year Cultivar PeakPaste Paste Set Back Amylose % Score Score 2003 L-206 225 160 286 6122.9 5 2.5 L-205 241 194 330 85 23.3 6 2.0 L-204 236 170 280 44 22.9 54.0 2004 L-206 260 152 297 37 22.8 5 2.5 L-205 288 192 365 76 24.9 6 2.0L-204 271 157 289 18 22.5 5 3.5 2005 L-206 272 171 312 40 22.3 5 2.5L-205 267 186 363 95 24.1 6 2.1 L-204 263 160 289 26 22.4 5 3.5 MeanL-206 252 161 298 46 22.7 5 2.5 L-205  265*  191*  353*  85* 24.1*  6*2.0 L-204 257 162 286  29* 22.6 5 3.7* *Over all year meanssignificantly different from L-206 at 0.05 probability level

Deposit Information

A deposit of the rice seed of this invention is maintained by theCalifornia Cooperative Rice Research Foundation, Inc., 955 Butte CityHighway, Biggs, Calif. 95917. Access to this deposit will be availableduring the pendency of this application to persons determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR 1.14 and 35 USC 122. Upon allowance of any claims in thisapplication, all restrictions on the availability to the public of thevariety will be irrevocably removed by affording access to a deposit ofat least 2,500 seeds of the same variety with the American Type CultureCollection, Manassas, Va.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A seed of rice cultivar L-206, wherein a representative sample ofseed of said cultivar was deposited under ATCC Accession No. PTA-______.2. A rice plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. A tissue culture of cells produced from the plant of claim2, wherein said cells of the tissue culture are produced from a plantpart selected from the group consisting of leaf, pollen, embryo,cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther,flower, stem, glumes and panicle.
 4. A protoplast produced from theplant of claim
 2. 5. A protoplast produced from the tissue culture ofclaim
 3. 6. A rice plant regenerated from the tissue culture of claim 3,wherein the plant has all the morphological and physiologicalcharacteristics of cultivar L-206.
 7. A method for producing an F1hybrid rice seed, wherein the method comprises crossing the plant ofclaim 2 with a different rice plant and harvesting the resultant F1hybrid rice seed.
 8. A hybrid rice seed produced by the method of claim7.
 9. A hybrid rice plant, or a part thereof, produced by growing saidhybrid seed of claim
 8. 10. A method of producing an herbicide resistantrice plant wherein the method comprises transforming the rice plant ofclaim 2 with a transgene wherein the transgene confers resistance to anherbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 11. An herbicide resistant rice plant produced by themethod of claim
 10. 12. A method of producing an insect resistant riceplant wherein the method comprises transforming the rice plant of claim2 with a transgene that confers insect resistance.
 13. An insectresistant rice plant produced by the method of claim
 12. 14. The riceplant of claim 13, wherein the transgene encodes a Bacillusthuringiensis endotoxin.
 15. A method of producing a disease resistantrice plant wherein the method comprises transforming the rice plant ofclaim 2 with a transgene that confers disease resistance.
 16. A diseaseresistant rice plant produced by the method of claim
 15. 17. A method ofproducing a rice plant with modified fatty acid metabolism or modifiedcarbohydrate metabolism wherein the method comprises transforming therice plant of claim 2 with a transgene encoding a protein selected fromthe group consisting of fructosyltransferase, levansucrase,alpha-amylase, invertase and starch branching enzyme or encoding anantisense of stearyl-ACP desaturase.
 18. A rice plant having modifiedfatty acid metabolism or modified carbohydrate metabolism produced bythe method of claim
 17. 19. A method of introducing a desired trait intorice cultivar L-206 wherein the method comprises: (a) crossing an L-206plant, wherein a representative sample of seed was deposited under ATCCAccession No. PTA-______, with a plant of another rice cultivar thatcomprises a desired trait to produce progeny plants wherein the desiredtrait is selected from the group consisting of male sterility, herbicideresistance, insect resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism and resistance to bacterial disease, fungaldisease or viral disease; (b) selecting one or more progeny plants thathave the desired trait to produce selected progeny plants; (c) crossingthe selected progeny plants with the L-206 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and the physiological and morphological characteristics ofrice cultivar L-206 to produce selected backcross progeny plants; and(e) repeating steps (c) and (d) three times to produce selected fourthor higher backcross progeny plants that comprise the desired trait andall of the physiological and morphological characteristics of ricecultivar L-206 as listed in Table
 1. 20. A plant produced by the methodof claim 19, wherein the plant has the desired trait and all of thephysiological and morphological characteristics of rice cultivar L-206as listed in Table
 1. 21. The plant of claim 20, wherein the desiredtrait is herbicide resistance and the resistance is conferred to anherbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 22. The plant of claim 20, wherein the desired trait isinsect resistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 23. The plant of claim 20,wherein the desired trait is modified fafty acid metabolism or modifiedcarbohydrate metabolism and said desired trait is conferred by a nucleicacid encoding a protein selected from the group consisting offructosyltransferase, levansucrase, alpha-amylase, invertase and starchbranching enzyme or encoding an antisense of stearyl-ACP desaturase.